Plant Physiology Preview. Published on August 17, 2016, as DOI:10.1104/pp.16.00660
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Reactive oxygen species tune root tropic responses
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Gat Krieger1†, Doron Shkolnik1†, Gad Miller2 and Hillel Fromm1*
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University, Tel Aviv 69978, Israel (G.K., D.S. and H.F.), 2 Mina and Everard Goodman Faculty of
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Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel (G.M.)
Department of Molecular Biology & Ecology of Plants, Faculty of Life Sciences, Tel Aviv
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Author contribution: G.K. and D.S. designed and performed experiments, analyzed the data and
wrote the manuscript. H.F. and G.M supervised experiments and participated in writing the
manuscript.
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Funding: This research was supported by the I-CORE Program of the Planning and Budgeting
Committee and The Israel Science Foundation (grant No 757/12).
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Summary: Biochemical, genetic and cellular evidence shows that ROS accelerates gravitropism but attenuates
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hydrotropism of Arabidopsis roots
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†
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*Corresponding author’s email: [email protected]
These authors (in alphabetical order) equally contributed to this manuscript.
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Copyright 2016 by the American Society of Plant Biologists
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Abstract
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The default growth pattern of primary roots of land plants is directed by gravity. However, roots
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possess the ability to sense and respond directionally to other chemical and physical stimuli,
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separately and in combination. Therefore, these root tropic responses must be antagonistic to
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gravitropism. The role of reactive oxygen species (ROS) in gravitropism of maize and
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Arabidopsis roots has been previously described. However, which cellular signals underlie the
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integration of the different environmental stimuli, which lead to an appropriate root tropic
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response, is currently unknown. In gravity-responding roots, we observed, by applying the ROS-
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sensitive fluorescent dye Dihydrorhodamine-123 and confocal microscopy, a transient
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asymmetric ROS distribution, higher at the concave side of the root. The asymmetry, detected at
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the distal elongation zone (DEZ), was built in the first two hours of the gravitropic response and
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dissipated after another two hours. In contrast, hydrotropically-responding roots show no
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transient asymmetric distribution of ROS. Decreasing ROS levels by applying the antioxidant
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ascorbate, or the ROS-generation inhibitor Diphenylene iodonium (DPI) attenuated gravitropism
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while enhancing hydrotropism. Arabidopsis mutants deficient in Ascorbate Peroxidase 1 (APX1)
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showed attenuated hydrotropic root bending. Mutants of the root-expressed NADPH oxidase
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RBOH C, but not rbohD, showed enhanced hydrotropism and less ROS in their roots apices
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(tested in tissue extracts with Amplex Red). Finally, hydrostimulation prior to gravistimulation
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attenuated the gravistimulated asymmetric ROS and auxin signals that are required for gravity-
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directed curvature. We suggest that ROS, presumably H2O2, function in tuning root tropic
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responses by promoting gravitropism and negatively regulating hydrotropism.
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Introduction
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Plants evolved the ability to sense and respond to various environmental stimuli in an integrated
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fashion. Due to their sessile nature, they respond to directional stimuli such as light, gravity,
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touch and moisture by directional organ growth (curvature), a phenomenon termed tropism.
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Experiments on coleoptiles conducted by Darwin in the 1880s revealed that in phototropism, the
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light stimulus is perceived by the tip, from which a signal is transmitted to the growing part
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(Darwin and Darwin, 1880). Darwin postulated that in a similar manner, the root tip perceives
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stimuli from the environment, including gravity and moisture, processes them and directs the
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growth movement, acting like “the brain of one of the lower animals” (Darwin and Darwin,
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1880). The transmitted signal in phototropism and gravitropism was later found to be a
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phytohormone, and its redistribution on opposite sides of the root or shoot was hypothesized to
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promote differential growth and bending of the organ (Went, 1926; Cholodny, 1927). Over the
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years, the phytohormone was characterized as indole-3-acetic acid (IAA, auxin) (Kögl et al.,
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1934; Thimann, 1935) and the 'Cholodny-Went' theory was demonstrated for gravitropism and
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phototropism (Rashotte et al., 2000; Friml et al., 2002). In addition to auxin, second messengers
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such as Ca2+, pH oscillations, Reactive Oxygen Species (ROS) and abscisic acid (ABA) were
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shown to play an essential role in gravitropism (Young and Evans, 1994; Fasano et al., 2001; Joo
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et al., 2001; Ponce et al., 2008). Auxin was shown to induce ROS accumulation during root
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gravitropism, where the gravitropic bending is ROS-dependent (Joo et al., 2001; Peer et al.,
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2013).
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ROS such as superoxide and hydrogen peroxide were initially considered toxic
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byproducts of aerobic respiration, but currently are known also for their essential role in myriad
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cellular and physiological processes in animals and plants (Mittler et al., 2011). ROS and
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antioxidants are essential components of plant cell growth (Foreman et al., 2003), cell cycle
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control and shoot apical meristem maintenance (Schippers et al., 2016) and play a crucial role in
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protein modification and cellular redox homeostasis (Foyer and Noctor, 2005). ROS function as
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signal molecules by mediating both biotic- (Sagi and Fluhr, 2006; Miller et al., 2009) and
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abiotic- (Kwak et al., 2003; Sharma and Dietz, 2009) stress responses. Joo et al. (2001) reported
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a transient increase in intracellular ROS concentrations early in the gravitropic response, at the
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concave side of maize roots, where auxin concentrations are higher. Indeed, this asymmetric
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ROS distribution is required for gravitropic bending, since maize roots treated with antioxidants,
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which act as ROS scavengers, showed reduced gravitropic root bending (Joo et al., 2001). The
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link between auxin and ROS production was later shown to involve the activation of NADPH
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oxidase, a major membrane-bound ROS generator, via a phosphatidylinositol 3-kinase-
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dependent pathway (Brightman et al., 1988; Joo et al., 2005; Peer et al., 2013). Peer et al. (2013)
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suggested that in gravitropism, ROS buffer auxin signaling by oxidizing the active auxin, IAA,
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to the non-active and non-transported form, oxIAA.
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Gravitropic-oriented growth is the default growth program of the plant, with shoots
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growing upwards and roots downwards. However, upon exposure to specific external stimuli, the
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plant overcomes its gravitropic growth program and bends towards or away from the source of
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the stimulus. For example, as roots respond to physical obstacles or water deficiency. The ability
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of roots to direct their growth towards environments of higher water potential was described by
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Darwin and even earlier, and was later defined as hydrotropism (Von Sachs, 1887; Jaffe et al.,
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1985; Eapen et al., 2005).
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In
Arabidopsis,
wild-type
(WT)
seedlings
respond
to
moisture
gradients
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(hydrostimulation) by bending their primary roots towards higher water potential. Upon
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hydrostimulation, amyloplasts, the starch-containing plastids in root-cap columella cells, which
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function as part of the gravity sensing system, are degraded within hours and recover upon water
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replenishment (Takahashi et al., 2003; Ponce et al., 2008; Nakayama et al., 2012). Moreover,
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mutants with a reduced response to gravity (pgm1) and to auxin (axr1 and axr2) exhibit higher
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responsiveness to hydrostimulation, manifested as accelerated bending compared to WT roots
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(Takahashi et al., 2002; Takahashi et al., 2003). Recently we have shown that hydrotropic root
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bending does not require auxin redistribution and is accelerated in the presence of auxin polar
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transport inhibitors and auxin-signaling antagonists (Shkolnik et al., 2016). These results reflect
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the competition, or interference, between root gravitropism and hydrotropism (Takahashi et al.,
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2009). However, which cellular signals participate in the integration of the different
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environmental stimuli that direct root tropic curvature is still poorly understood. Here we sought
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to assess the potential role of ROS in regulating hydrotropism and gravitropism in Arabidopsis
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roots.
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Results
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Different spatial and temporal ROS patterns occur in roots in response to hydrostimulation
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and gravistimulation
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In order to investigate the role of ROS signals in tropic responses we first assessed the spatial
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distribution of ROS in Arabidopsis roots responding to gravitropic stimulation. WT Arabidopsis
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seedlings grown vertically on agar-based medium (Materials and Methods) were gravistimulated
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by a 90º rotation, and monitored for their ROS distribution by applying Dihydrorhodamine-123
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(DHR), a rhodamine-based fluorescent probe mostly sensitive to H2O2 (Gomes et al., 2005) that
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is often used in monitoring intracellular, cytosolic ROS (Royall and Ischiropoulos, 1993; Crow,
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1997; Douda et al., 2015). DHR staining was detected in the columella, lateral root cap,
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epidermal layer of elongation zone (EZ) and the vasculature, and was weaker at the meristematic
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zone (Fig.1). This pattern is similar to previously reported staining patterns obtained by H2O2-
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specific dyes in primary roots of Arabidopsis (Dunand et al., 2007; Tsukagoshi et al., 2010; Chen
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and Umeda, 2015) and of other plant species (Ivanchenko et al., 2013; Xu et al., 2015). One to
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two hours post gravistimulation, a ROS asymmetric distribution, higher at the concave (bottom
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side of the root) was apparent in the epidermal layer of the distal elongation zone (DEZ), where
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the bending initiates (Fig.1 A). The asymmetric ROS distribution dissipated after another two
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hours (Fig.1 A, D), in accordance with previous reports (Joo et al., 2001; Peer et al., 2013).
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To study ROS dynamics during hydrotropic growth, WT seedlings were introduced into a
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moisture gradient in a closed CaCl2-containing chamber (herein referred to as the CaCl2 / dry
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chamber) as previously described (Takahashi et al., 2002; Kobayashi et al., 2007; Shkolnik et al.,
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2016). Under this system root bending upon hydrostimulation initiates at a region more distant
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from the root tip compared to root bending by gravitropism. The distances of curvature from the
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root tip for hydrotropism and gravitropism were 601.2 ± 18.1 μm and 365.1 ± 13.1 μm,
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respectively (mean ± SE), 2 h post stimulation (n=29). We therefore designated the region of
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gravitropic bending initiation as the distal elongation zone (DEZ) and the region of hydrotropic
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bending initiation as the central elongation zone (CEZ), in accordance with previous definitions
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(Fasano et al., 2001; Massa and Gilroy, 2003). Furthermore, during the hydrotropic response, the
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root tip keeps facing downwards in response to gravity, where a slight curvature is detected in
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the DEZ (Fig.1 B, 1, 2 and 4 hours, concave side is indicated). Interestingly, during hydrotropic
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growth, ROS do not form an asymmetric distribution pattern at the DEZ, in contrast to the
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gravity-induced ROS asymmetric distribution (Fig.1 B, D). However, asymmetric distribution of
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ROS appears at the CEZ, where the hydrotropic root curvature takes place and detected ROS
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levels are lower (Fig.1 B, D). This unequal distribution of ROS appears, however, also in roots
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that were subjected to non-hydrostimulating conditions (obtained by adding distilled water to the
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bottom the chamber), which do not undergo hydrotropic bending (Fig.1 C). Under these
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experimental conditions, a higher ROS level was measured at the side of the root facing the agar
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medium (Fig.1 C, arrowhead). The CEZ-located asymmetric distribution is not dynamic, and is
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maintained throughout the first four hours of the hydrotropic response without a significant
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change in the ratio level between the two sides of the root (Fig.1 B, D). We suspected that this
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asymmetric distribution of ROS may be caused by the mechanical tension formed as the root
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bends around the agar bed. To further test this, we used the split-agar / sorbitol system (Materials
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and Methods) for assessing ROS distribution during hydrotropism. In this experimental system,
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no asymmetric ROS distribution could be detected in response to hydrostimulation in the DEZ or
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CEZ (Fig.1 E, D). Moreover, we detected no changes in the overall intensity of DHR
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fluorescence at the indicated time points in both hydrostimulated and gravistimulated roots
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(Supplemental Fig.S1). Collectively, these results depict distinct dynamics and spatial patterns of
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ROS distribution during gravitropic and hydrotropic responses, which may imply different roles
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of ROS in these tropic responses. We note that strong DHR fluorescence is detected in the root
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vasculature above the CEZ at all time points, similar to previous reports (Tsukagoshi et al., 2010;
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Chen and Umeda, 2015).
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ROS tune root tropic responses
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To assess the possible role of ROS in hydrotropism compared to gravitropism, we tested whether
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ROS scavenging molecules or ROS-generation inhibitors affect hydrotropic growth. As
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described previously, the antioxidant ascorbic acid (ascorbate) has an inhibitory effect on root
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gravitropism (Joo et al., 2001; Peer et al., 2013). Indeed, our results show gravitropic bending
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inhibition in the presence of 1 mM ascorbate, a concentration that we found to significantly
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reduce ROS level at the root tip (Supplemental Fig.S2). Root curvature in control conditions was
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64.9 ± 2.6 degrees, whereas in the presence of ascorbate only 49.1 ± 5.2 degrees (mean ± SE) 8 h
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post gravistimulation (P=0.011, Student's t test for independent measurements), without
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differences in root growth rates (Supplemental Fig.S3). In contrast, application of 1 mM
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ascorbate accelerated hydrotropic root bending. Root curvature in the CaCl2 / dry chamber was
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27.2 ± 2.6 degrees in control conditions whereas in the presence of ascorbate curvature was 39.3
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± 3.5 degrees (mean ± SE) 2 h post hydrostimulation (P=0.01, Student's t test for independent
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measurements), and reduced root growth rate by 29.4% (Fig.2 A, B). The same trend was
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apparent when 1 mM of the antioxidant N-Acetyl-Cysteine was applied (not shown).
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To further study the effect of ascorbate metabolism on hydrotropism we tested mutants
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deficient in the most abundant cytosolic ascorbate peroxidase, Ascorbate Peroxidase 1 (APX1)
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(Davletova et al., 2005). apx1-2 seedlings exhibited attenuated hydrotropic bending compared to
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WT. Root curvature in the CaCl2 / dry chamber of WT was 72.0 ± 2.8 degrees whereas that of
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apx1-2 was 55.8 ± 3.5 degrees (mean ± SE) 5 h post hydrostimulation (P=9.6 ∗ 10 , Student's t
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test for independent measurements), with no differences in their growth rates (Fig.2 C, D). These
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results were reproduced using the split-agar / sorbitol system in which the ascorbate was
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supplemented to the sorbitol agar slice, allowing diffusion of the chemicals towards the root tip
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so that the exposure to ascorbate occurs while a water potential gradient is formed (Takahashi et
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al., 2002; Antoni et al., 2016) (Supplemental Fig.S4 A, B). These data strongly suggest that the
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reduced ability to scavenge cytosolic H2O2 inhibited root hydrotropic bending. Unlike ascorbate-
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treated seedlings, gravitropic bending was not impaired or promoted in the apx1-2 mutant
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(supplemental Fig.S7).
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ROS generation by NADPH oxidase has opposite effects on different root tropic responses
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To further study the roles of ROS in root tropisms, we tested the effects of diphenylene iodonium
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(DPI), an inhibitor of NADPH oxidase and other flavin-containing enzymes (Foreman et al.,
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2003), on hydrotropic- and gravitropic-bending kinetics and the corresponding ROS distribution
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patterns in primary roots. NADPH oxidase is a plasma membrane-bound enzyme that produces
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superoxide (O2•–) to the apoplast (Sagi and Fluhr, 2006). Superoxide is rapidly converted to
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H2O2, which may enter the cell passively or through aquaporins (Miller et al., 2010; Mittler et
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al., 2011). Application of DPI accelerated hydrotropic root bending but attenuated gravitropic
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root bending (Fig.3). In response to hydrostimulation, root bending was accelerated in the
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presence of DPI, showing 86.3 ± 2.1 degrees curvature (mean ± SE) in the CaCl2 / dry chamber
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after only 4 h, even though root growth rate was inhibited by 65.3% (Fig.3). This result was
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reproduced using the split-agar / sorbitol system (Supplemental Fig.S4 A).
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Fluorescent ROS staining of DPI-treated roots revealed elimination of ROS from the
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epidermal layer of the EZ and further along the root, where ROS at the outer layers (epidermis
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and cortex) seemed to drop down and the remaining ROS appeared in the vasculature and its
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surrounding layers (Fig.4 A, B). ROS elimination at the outer root cell layers was previously
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described for hydroxyphenyl fluorescein (HPF)-staining upon DPI treatment (Dunand et al.,
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2007). Along with decreased fluorescence at the EZ, we detected an increase of DHR
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fluorescence intensity at the meristematic zone of DPI-treated roots (Fig.4). Dunand et al. (2007)
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used nitroblue tetrazolium (NBT) for assessing extracellular O2•– levels in Arabidopsis root tips,
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and detected a decrease in NBT intensity upon DPI treatment. Since the DHR probe is mostly
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sensitive to cytosolic H2O2 (Gomes et al., 2005), our results do not contradict previously reported
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results.
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Gravistimulated seedlings that were pre-treated for 2 h with DPI showed less ROS
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accumulation and consequently no ROS asymmetric distribution in the epidermal layer of the
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EZ, resulting in a delayed gravitropic response (Fig.4 C). Similarly, seedlings that were
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hydrostimulated in the presence of DPI showed elimination of ROS from the epidermal layer at
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the bending region, which became more proximal to the root tip (Fig.4 D). Interestingly, the
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gravity-directed curvature of the root tip, which occurs during hydrotropic root bending,
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appeared to be attenuated in ascorbate- and DPI-treated seedlings (Fig.2 A, Fig.4 D). This
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finding demonstrates again the negative effect of ROS elimination on root gravitropism, also in
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combination with a hydrotropic response.
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Hydrotropism is affected by root NADPH oxidase
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To further assess the inhibitory effect of ROS generation by NADPH oxidase on root
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hydrotropism we tested transposon-insertion mutants of the plant NADPH oxidase - RBOH
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(Respiratory Burst Oxidase Homolog) gene family, which consists of 10 members in
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Arabidopsis. These can be divided into three classes based on their tissue-specificity: RBOH D
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and F are highly expressed throughout the plant, RBOH A-G and I are expressed mostly in roots,
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and RBOH H and J express specifically in pollen (Sagi and Fluhr, 2006). RBOH C has been
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intensively studied, and its activity in ROS production in trichoblasts is essential for root hair
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elongation and mechanosensing (Foreman et al., 2003; Monshausen et al., 2009). It is expressed
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in trichoblasts and in the epidermal layer of the EZ (Foreman et al., 2003), though its role in the
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EZ is still unclear (Monshausen et al., 2009). When hydrostimulated in the CaCl2 / dry chamber
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or in the split-agar / sorbitol systems, rbohC seedlings exhibited accelerated hydrotropic bending.
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Measured in the CaCl2 / dry chamber, root curvature in WT was 46.4 ± 3.1 degrees compared to
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64.2 ± 3.5 degrees in rbohC (mean ± SE) 2 h post hydrostimulation (P=5.1 ∗ 10 , student's t
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test for independent measurements) with no difference in growth rate compared to WT (Fig.5 A,
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B; Supplemental Fig.S4 C; Supplemental movie 1). We then examined the hydrotropic response
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of seedlings deficient in RBOH D, which has the highest expression levels among the RBOHs.
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RBOH D is expressed in all plant tissues but mainly in stems and leaves and is known as a key
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factor in ROS systemic signaling (Sagi and Fluhr, 2006; Miller et al., 2009; Suzuki et al., 2011).
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Interestingly, rbohD seedlings did not exhibit significantly-different hydrotropic bending kinetics
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or root growth rates compared to WT (Fig. 5 A, B; Supplemental Fig.S4 C; Supplemental movie
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2). DHR staining revealed no significant difference in ROS spatial patterns in gravistimulated
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nor hydrostimulated (using the CaCl2 / dry chamber or split-agar / sorbitol system) roots of the
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RBOH mutants, compared to WT (Supplemental Fig.S5-S8). Therefore, to better characterize
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endogenous ROS levels in root tissues of wt and rbohc and rbohd mutants, we applied Amplex
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red for determination of H2O2 content in tissue extracts (Materials and Methods). When
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examining extracts from whole seedlings, we observed a 68% and 77% reduction in H2O2 levels
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in rbohD and rbohC, respectively, compared to WT (Fig.5 D). We then examined extracts from
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excised root apices (1-2 mm from tip) and observed a relatively similar H2O2 content in WT and
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rbohD roots, while rbohC mutants showed a 57% reduction in H2O2 content compared to WT
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(Fig.5 C). These results are consistent with the tissue-specific expression pattern of the two
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RBOHs, as RBOH C is highly expressed in roots, while RBOH D is not (Sagi and Fluhr, 2006)
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and with the accelerated hydrotropic phenotype of rbohC compared to rbohD and wt. Their
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different expression patterns could also be visualized in the high-resolution spatiotemporal map
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(Brady et al., 2007) of the eFP browser (Winter et al., 2007).
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The acceleration in hydrotropic root bending of rbohC is however weaker compared with
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that of DPI-treated WT seedlings (measured in the CaCl2 / dry chamber, root curvature in rbohC
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was 75.41±2.19 degrees and root curvature of DPI treated seedlings was 86.31±2.11 degrees
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after 4 h of hydrostimulation, while WT and DMSO-treated WT roots exhibited 63.27±2.38 and
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62.67±3.17 degrees in that time, respectively). These results may indicate partial functional
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redundancy with other root-expressed RBOHs, or involvement of other DPI-sensitive enzymes in
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this tropic growth. When treated with DPI, rbohC roots presented the same hydrotropic bending
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kinetics as WT roots (not shown). Unlike DPI-treated seedlings, RBOH C- and RBOH D-
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deficient mutants did not show inhibition or acceleration in their gravitropic growth
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(Supplemental Fig.S8) nor weakened gravity-directed curvature of the root tip during
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hydrotropic growth (Fig.5) and gravitropic ROS asymmetric distribution as in WT
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(Supplemental Fig.S7). These results may be explained by functional redundancy between the
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root-expressed RBOH family members, as well as by compensation of ROS signaling by
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mechanisms involved specifically in gravitropism.
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Hydrotrostimulation attenuates the gravitropic ROS and auxin signals
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In order to test a possible direct link between hydrotropism and gravitropism through ROS, we
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challenged WT seedlings with combined stimuli using the split-agar / sorbitol method (Fig.6 A).
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The split-agar system allows slow and controlled exposure of the root tips to increasing osmotic
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pressure, and by rotation of the chamber allows changes in the gravity vector (Fig.6 A). After 0-2
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h of hydrostimulation, 1 h of gravistimulation induced a clear asymmetric ROS distribution at
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the bending EZ. After 3 h of hydrostimulation, 1 h of gravistimulation generated a weak
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asymmetric ROS distribution (Fig.6 B, C). Strikingly, following 4 h of hydrostimulation, 1 h of
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gravistimulation failed to generate an asymmetric ROS distribution, and gravity-directed root
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bending was not observed (Fig.6 B, C). These results indicate that as the osmotic stress stimulus
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increases and promotes hydrotropic curvature, gravistimulation is not sufficient to evoke typical
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ROS asymmetric distribution, and growth towards higher water potential is favorable. Indeed,
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with increasing hydrostimulation time from 0 to 4 hr prior to gravistimulation, gravitropic
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curvature decreased (Fig.6 D). Four hrs of hydrostimulation prevented gravitropic curvature as
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roots responded only to the hydrotropic stimulus (depicted as a negative curvature angle in Fig.6
278
D).
279
Subsequently, in order to assess whether the attenuation of the ROS signal of
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gravistimulated roots following hydrostimulation is associated with the attenuation of auxin
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distribution, roots of DII-VENUS-expressing transgenic seedlings (Brunoud et al., 2012) were
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gravistimulated for 1 h following exposure to an osmotic gradient for 0, 2 or 4 h (Supplemental
283
Fig.S9). With this auxin reporter, lower levels of DII-VENUS fluorescence indicate higher levels
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of auxin. In agreement with the ROS signal dynamics, we observed asymmetric auxin
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distribution in the lower part of the root tip (concave) in roots that were gravistimulated with no
286
prior hydrostimulation, or following 2 h of hydrostimulation (Supplemental Fig.S9), as
287
previously
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hydrostimulation for 4 h prior to gravistimulation impaired the generation of an auxin gradient
289
across the root tip (Supplemental Fig.S9). Based on the known relationship between auxin and
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ROS in gravistimulation, these results may suggest that hydrotropic stimulation attenuates the
demonstrated
in
graviresponding
roots
(Band
et
al.,
2012).
However,
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gravitropic ROS signal through the interruption of auxin distribution. However, we cannot
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exclude the possibility that hydrostimulation attenuates gravistimulated ROS and auxin
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distribution through independent signaling pathways that are yet to be elucidated.
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Discussion
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In order to perform hydrotropic bending, a root must overcome its gravity-directed growth
296
(Eapen et al., 2005; Takahashi et al., 2009). Our results suggest opposite roles for ROS in
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hydrotropic and gravitropic growth behaviors. When treated with ascorbate, an antioxidant, or
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DPI, an inhibitor of NADPH oxidase and other flavin-containing enzymes (Foreman et al.,
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2003), Arabidopsis primary roots exhibit opposite changes in their bending kinetics in response
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to the different stimulations, namely, delay in gravitropism and acceleration in hydrotropism
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(Fig.2, 3 ,Supplemental Fig. S3 and Supplemental Fig.S4). The antagonism between these two
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responses was shown previously for the agravitropic pea mutant (ageotropum), whose lack of
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gravity response contributes to its hydrotropic responsiveness (Takahashi and Suge, 1991).
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Amyloplast degradation at early stages of a hydrotropic response may also be a mechanism by
305
which the root eliminates its sense of gravity in order to perform non-gravitropic growth
306
(Takahashi et al., 2003; Ponce et al., 2008). When examining the ROS and auxin patterns in
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response to combined stimuli by first applying hydrostimulation and afterwards applying both
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hydro- and gravistimulation, we observed a reduction in gravity-directed ROS-asymmetry and
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auxin-gradient when the duration of hydrostimulation is increased (Fig.6, Supplemental Fig.S9).
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We therefore conclude that during hydrotropic growth, the root actively attenuates gravitropic
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auxin and ROS signaling to overcome gravitropic growth.
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In gravitropism, auxin is required for ROS production (Joo et al., 2005; Peer et al., 2013).
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In contrast, neither auxin redistribution nor auxin signaling are required for hydrotropic bending
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(Shkolnik et al., 2016). Moreover, inhibition of polar auxin transport or Transport Inhibitor
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Response (TIR)-dependent signaling accelerate hydrotropism (Shkolnik et al., 2016). Consistent
316
with these observations, asymmetric distribution of ROS was not detected in the DEZ during
317
hydrotropism. In gravitropism, however, both an auxin gradient at the lateral root cap, and ROS
318
asymmetric distribution at the DEZ are formed transiently. Collectively, these results
319
demonstrate the antagonism between hydro- and gravitropism with respect to auxin- and ROS-
320
signaling.
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321
Asymmetric ROS distribution was however observed in the CEZ of hydrostimulated
322
roots in the CaCl2 / dry chamber system, and its asymmetry ratio level has not changed during
323
the measured time points (Fig.1 B, D). This asymmetric pattern, i.e., higher ROS levels at the
324
side of the root that is in contact with the agar medium, was also present in roots that were
325
exposed to non-hydrostimulating conditions and do not perform hydrotropic bending (Fig.1 C,
326
D). Therefore, this non-transient unequal distribution of ROS in the CEZ may be a result of
327
mechanosensing-induced ROS (Monshausen et al., 2009) at the region where the root detaches
328
from the agar medium. Indeed, no ROS asymmetry was observed in roots exposed to a water-
329
potential gradient in the split-agar / sorbitol system (Fig.1 E,D), where the root does not
330
encounter mechanical tension by the agar due to bending. Therefore it is clear that hydrotropism
331
does not involve asymmetric distribution of ROS. Yet, it attenuates gravity-directed asymmetric
332
ROS distribution.
333
In addition to their roles as intracellular signaling molecules, ROS function in several
334
apoplastic processes, including cell wall rigidification that is thought to restrict cell elongation
335
(Hohl et al., 1995; Monshausen et al., 2007). It is tempting to hypothesize that in gravitropism,
336
the higher levels of ROS in the concave side of the root promote root bending by inhibition of
337
cell elongation at this side. However, this hypothesis fails to explain the opposite effects of
338
antioxidants and ROS-generator inhibitors on gravi- and hydrotropism, as differential cell
339
elongation is needed in both cases.
340
In this study, we show that ROS, presumably cytosolic H2O2 in the epidermal layer of the
341
root EZ, negatively regulate hydrotropic bending. The activity of RBOH C was characterized as
342
essential for this process, since rbohC mutants showed accelerated hydrotropic root bending and
343
lower levels of H2O2 in the root apex (Fig.5). This, however, does not exclude the possible
344
contribution of other root-expressed RBOHs or other flavin-containing enzymes to the process.
345
The localization of ROS-generating enzymes of the RBOH family has substantial effects on the
346
tissue-specific ROS levels and the consequent hydrotropic root curvature, as it appears that in
347
mutants deficient in RBOH D, which is expressed throughout the plant but mostly in leaves and
348
stems (Suzuki et al., 2011) ROS levels in the root apex and hydrotropic curvature were similar to
349
those of WT (Fig.5, Supplemental Fig.S3). As for ROS scavenging enzymes, we detected a weak
350
hydrotropic root bending in apx1-2 mutants (Fig.2, Supplemental Fig.S3), which lack the
351
function of the abundant cytosolic H2O2-scavenging enzyme APX1 and are thus expected to
352
accumulate higher H2O2 levels in all plant tissues. Peroxidases were shown to play an important
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353
role in root development and growth control (Dunand et al., 2007) by modifying O2•– to H2O2 at
354
the transition-to-elongation zone (Tsukagoshi et al., 2010). Our observations are consistent with
355
this ROS type-specific accumulation pattern, and add a new aspect to the role of H2O2 at the root
356
EZ.
357
The phytohormone abscisic acid (ABA) was previously reported as a positive regulator of
358
root hydrotropism. Arabidopsis mutants deficient in ABA-sensitivity (abi2-1) and ABA-
359
biosynthesis (aba1-1) were reported as less responsive to hydrostimulation, whereas ABA
360
treatment rescued the delayed hydrotropic phenotype of aba1-1 (Takahashi et al., 2002). ABA-
361
signaling involves the activation of Pyrabactin Resistance/PYR1-like (PYR/PYL) receptors that
362
mediate the inhibition of clade A phosphatases type 2C (PP2C), which are negative regulators of
363
the pathway (Antoni et al., 2013). The involvement of this pathway in root hydrotropism was
364
demonstrated recently, as a pp2c-quadruple mutant exhibited an ABA-hypersensitive phenotype
365
and consequently enhanced hydrotropic response, while a mutant deficient in six PYR/PYL
366
receptors exhibited insensitivity to ABA treatment and to hydrotropic stimulation (Antoni et al.,
367
2013). Since ABA was shown to induce stomata closure through the activation of the NADPH
368
oxidases RBOH D and RBOH F (Kwak et al., 2003), it is tempting to hypothesize that ABA
369
activates ROS production in root-expressed NADPH oxidases during hydrotropic growth. A
370
candidate mediator for this process may be PYL8, since PYL8-deficient mutants (pyl8-1 and
371
pyl8-2) exhibited a non-redundant ABA-insensitive root growth when treated with ABA, and
372
transcriptional fusion of PYL8 (ProPYL8:GUS) revealed its expression in the stele, columella,
373
lateral root cap and root epidermis cells (Antoni et al., 2013). The latter expression region
374
overlaps with that of RBOH C (Foreman et al., 2003). However, distinguished from their role in
375
stomata closure, ROS negatively regulate hydrotropism and thus may function in a negative
376
feedback to ABA signaling. Antagonism between ROS and ABA also appears in seed
377
germination, as H2O2 breaks ABA-induced seed dormancy in several plant species (Sarath et al.,
378
2007).
379
In the context of integration of environmental stimuli by the root tip (Darwin and Darwin,
380
1880), we suggest that ROS, presumably cytosolic hydrogen peroxide, fine tune root tropic
381
responses by acting as positive regulators of gravitropism and as negative regulators of
382
hydrotropism. Root hydrotropism and gravitropism differ in several aspects, such as the time of
383
response (Eapen et al., 2005), the region of bending initiation (reported in this study), the
384
involvement of auxin (Kaneyasu et al., 2007; Shkolnik et al., 2016) and the effect of ROS on the
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385
response kinetics. In order to elucidate the effects of ROS on tropic responses, their downstream
386
effectors in gravitropism and hydrotropism need to be characterized.
387
Materials and Methods
388
Plant material and growth conditions
389
Wild type Arabidopsis thaliana (Col-0) and T-DNA/Transposon insertion mutants: rbohC (rhd2),
390
rbohD (Miller et al., 2009) and apx1-2 (SALK_000249) (Suzuki et al., 2013) were used in this
391
research. For vapor sterilization, seeds were put inside a desiccator next to a glass beaker
392
containing 25 ml water, 75 ml bleach and 5 ml HCl for 2 h. Sterilized seeds were sown on 12 x
393
12 cm squared Petri dishes, containing 2.2 gr/L Nitsch & Nitsch medium (Duchefa Biochemie
394
B.V., Haarlem, the Netherlands) titrated to pH 5.8, 0.5 % (w/v) sucrose supplemented with 1 %
395
(w/v) plant agar (Duchefa) and vernalized for one day in 4º C in dark. Plates were put vertically
396
in a growth chamber at 22º C and day light (100 µE m-2 sec-1) under 16/8 light/dark photoperiod.
397
The root hair-deficient phenotype of rbohC was observed when grown on pH 5-titrated growth
398
medium. Treatments with 10 µM DPI (Diphenyleneiodonium chloride, Sigma) dissolved in
399
Dimethyl Sulfoxide (DMSO), 1 mM Sodium Ascorbate (Sigma) dissolved in distilled water and
400
1 mM N-acetyl-cysteine (Acros organics) dissolved in distilled water were performed by
401
applying these chemicals in the agar medium. Ascorbate treatment for DHR staining was
402
performed by transferring seedlings onto 1 mm Whatman filter paper 0.25 X Murashige and
403
Skoog medium (MS) (Murashige and Skoog, 1962) and the indicated ascorbate concentrations.
404
Hydrotropic stimulation assays
405
A CaCl2 dry chamber was designed based on a previously described system (Takahashi et al.,
406
2002; Kobayashi et al., 2007; Shkolnik et al., 2016) with the following modifications: Plates
407
were prepared as described in ‘Plant material and growth conditions’ with or without
408
supplemented chemicals, as indicated. The medium was cut 6 cm from the bottom and 5-7 day-
409
old seedlings were transferred to the cut medium, such that approximately 0.2 mm of the primary
410
root tip was bolting from the agar into air. Twelve ml of 40 % CaCl2 (w/v) (Duchefa) were put at
411
the bottom of the plate, which was then closed, sealed with Parafilm and placed vertically under
412
30 µE m-2 sec-1 white light. As control, non-hydrostimulating conditions were achieved by
413
adding 20 ml of distilled water to the bottom of the plate. In this system, the roots were exposed
414
to the supplemented chemical at the beginning of the experiment. Hydrostimulation was
415
performed also using the previously described split-agar method (Takahashi et al., 2002; Antoni
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416
et al., 2016). Ascorbate, DPI or DMSO (control) were added directly to the sorbitol containing
417
gel slice. Root tips were imaged at indicated time points using Nikon D7100 camera equipped
418
with AF-S DX Micro NIKKOR 85 mm f/3.5G ED VR lens (Nikon, Tokyo, Japan). For root
419
curvature measurements and supplemental movies of the humidity-gradient system, plates were
420
faced ~45 º to the lens, and multiple photos with changing focus were obtained using Helicon
421
remote software, and stacked using Helicon focus software (www.heliconsoft.com). Root
422
curvature and growth were analyzed using ImageJ software 1.48V (Wayne Rasband, NIH, USA).
423
Gravitropic stimulation assay
424
Five to seven-day-old seedlings were transferred to a standard medium, or ascorbate containing
425
medium, following one hour of acclimation at original growth orientation before the plates were
426
90º rotated. For DPI treatment, seedlings were pre-treated in DMSO or 10 µM DPI-containing
427
media for 2 h, then transferred to another plate containing standard medium, followed by 30 min
428
acclimation at the original growth orientation before the plates were rotated by 90º.
429
Confocal microscopy
430
For ROS detection, seedlings were immersed in 86.5 µM [0.003% (w/v)] Dihydrorhodamine-123
431
(Sigma) dissolved in Phosphate Buffer Saline (PBS x 1, pH 7.4) for 2 or 5 min, after hydrotropic
432
or gravitropic stimulation assays. Fluorescent signals in roots were imaged with a Zeiss LSM
433
780 laser spectral scanning confocal microscope (Zeiss, http://corporate.zeiss.com), with a 10X
434
air (EC Plan-Neofluar 10x/0.30 M27) objective. Acquisition parameters were as follows: master
435
gain was always set between 670 and 720, with a digital gain of 1, excitation at 488 nm (2%) and
436
emission at 519-560 nm. Signal intensity was quantified as mean grey value using ImageJ
437
software. Confocal images were pseudo-colored using the RGB look-up table of the ZEN
438
software, for easier detection of the fluorescent signal distribution in the root. Imaging of DII-
439
VENUS expressing roots was performed as previously described (Shkolnik et al., 2016).
440
Determination of H2O2 in tissue extracts
441
Whole seedlings (n = 20 seedlings) and root apices (1-2 mm from root tip, n = 60 seedlings)
442
were frozen in liquid nitrogen and homogenized in Phosphate Buffer Saline (PBS x 1, pH 7.4)
443
(600 µl for whole seedlings and 150 µl for root apices), centrifuged in 10,000 g for 5 min in 4° C
444
and the supernatant was used as the tissue extract. H2O2 levels in the extracts were measured
445
using the Amplex red assay kit (Molecular Probes, Invitrogen) according to the manufacturer’s
446
protocol, with 3 biological repeats and two technical replicates. Samples were measured with a
15
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447
Synergy HT fluorescence plate reader (BioTek) using 530/590 nm excitation/emission filters.
448
Protein levels in the extracts were determined using the Bradford reagent (Bio-Rad). The
449
absorbance was read in the same plate reader using a 595 nm filter. Fluorescence reads were then
450
normalized to the protein amount.
451
Statistical analysis
452
Results were analyzed using MS Excel ToolPak and R version 3.1.1.
453
454
Supplemental materials
455
Figure S1: Relative DHR fluorescence intensity in gravistimulated and hydrostimulated roots.
456
Figure S2: ROS level is reduced by ascorbate.
457
Figure S3: The antioxidant ascorbate impedes root gravitropic response.
458
Figure S4: Hydrostimulation using the split-agar / sorbitol method.
459
Figure S5: ROS distribution during hydrotropic growth in WT, rbohC and rbohD mutants.
460
Figure S6: ROS distribution in hydrostimulated WT, rbohC and rbohD mutants using the split-
461
agar / sorbitol system.
462
Figure S7: ROS distribution in gravistimulated WT, apx1-2, rbhoC and rbhoD mutants.
463
Figure S8: rbohC and rbohD exhibit normal gravitropic growth compared to WT.
464
Figure S9: Auxin distribution in gravistimulated root tips with or without prior
465
hydrostimulation.
466
Video movie-1: Hydrotropism of rbohC mutant compared to wt.
467
Video Movie-2: Hydrotropism of rbohD mutant compared to wt.
468
469
Acknowledgements
470
This research was supported by the I-CORE Program of the Planning and Budgeting Committee
471
and The Israel Science Foundation (grant No 757/12). We thank Professor Robert Fluhr for
16
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
472
critical reading of the manuscript, and lab members Yosef Fichman and Roye Nuriel for helpful
473
suggestions.
474
Figure Legends
475
476
Figure 1: ROS spatial and temporal distribution patterns during root gravitropism and
477
hydrotropism. A, B, C and E) Confocal microscopy of 5-day old seedlings stained with
478
Dihydrorhodamin-123 (DHR), a ROS-sensitive fluorescent dye. Images were pseudo-colored,
479
red indicates higher ROS-dependent fluorescence intensity. Scale bars, 100 µm. DEZ, Distal
480
Elongation Zone, CEZ, Central Elongation Zone (designated according to Fasano et al., 2001).
481
White lines next to the root mark defined root zones. g represents gravity vector, Ψ represents
482
water potential gradient. Concave and convex sides of the root are indicated. Arrowheads point
483
to regions where the fluorescence signal distributes unevenly between the two sides of the root.
484
A) Under gravistimulation, an asymmetric distribution of ROS was apparent 2 h post stimulation
485
and dissipated after another 2 h. This asymmetry was detected at the DEZ where higher ROS
486
levels were observed at the concave side of the root. B) Under hydrostimulation, ROS distribute
487
asymmetrically at the CEZ however maintain symmetric distribution at the DEZ. C) The
488
asymmetric ROS pattern at the CEZ was also observed in roots that were exposed to non-
489
hydrostimulating conditions and do not bend hydrotropically. The higher ROS level was
490
observed at the side that is in contact with the agar medium. D) Quantification of DHR
491
fluorescence, measured at the epidermal layer in two regions of the root EZ (in the DEZ of
492
gravistimulated roots and in the DEZ and CEZ of hydrostimulated roots). The data is presented
493
as the ratio between the signal at the concave and the convex sides of the root. Error bars
494
represent mean ± SE (3 biological independent experiments, 14<n<23). **p < 0.01, Student's t-
495
test versus start time. E) Roots were hydrostimulated for the indicated times using the split-agar /
496
sorbitol system. F) Quantification of DHR fluorescence, measured at the DEZ epidermal layer
497
(200 µm above apex) and CEZ (600 µm above apex). The data is presented as the ratio between
498
the signal at the concave and the convex sides of the root. Error bars represent mean ± SE (3
499
biological independent experiments, n=20). No significant difference was found among different
500
hydrostimulation times (Tukey-HSD post hoc-test (P < 0.05)).
501
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502
Figure 2: Ascorbate accelerates root hydrotropic growth, and a mutant deficient in APX1 shows
503
attenuated hydrotropic bending. A) Seedlings performing hydrotropic bending 2.5 h post
504
hydrostimulation in the presence or absence of 1 mM sodium ascorbate. In both A and C) g
505
represents gravity vector, Ψ represents water potential gradient, Scale bar, 1 mm. B) Root
506
curvature kinetics and growth rate of ascorbate-treated hydrostimulated seedlings. Root
507
curvature was measured at 1 h interval for 7 h following hydrostimulation. Root growth rate was
508
determined by measuring the length at the beginning and at the end of the experiment. Error bars
509
represent mean ± SE (3 biological independent experiments, 10 seedlings each). *p <0.05,
510
Student’s t-test for independent measurements. C) Root hydrotropic bending of WT and apx1-2,
511
5 h post hydrostimulation. D) Root curvature kinetics and growth rate of apx1-2 and WT
512
hydrostimulated seedlings. Root curvature and root growth rate were measured as described in
513
B).
514
515
Figure 3: Application of DPI, an NADPH oxidase inhibitor, accelerates hydrotropism while
516
delaying gravitropism. A) Application of 10 µM Diphenyleneiodonium (DPI) to the growing
517
medium promotes hydrotropic curvature (first two left panels), and impedes gravitropic
518
curvature (two right panels). Images were taken 2 h post hydrostimulation (scale bar, 1 mm) and
519
12 h post gravistimulation (scale bar, 5 mm). g represents gravity vector, Ψ represents water
520
potential gradient. B) Root curvature was measured at 1 h interval for 6 h following
521
hydrostimulation and at 2 h interval for 12 h following gravistimulation. Error bars represent
522
mean ± SE (3 biological independent experiments, 10 seedlings each). C) DPI inhibits root
523
growth in both physiological assays. Root growth rate was determined by measuring length at
524
the beginning and at the end of the experiment. Error bars represent mean ± SE (3 biological
525
independent experiments, 10 seedlings each). **p<0.01, t-test for independent measurements.
526
Figure 4: DPI eliminates ROS levels at the epidermal layer of the root elongation zone and
527
elevates ROS levels at the meristematic zone. A, C and D) DHR fluorescence (in A, over bright
528
field, in C and D, fluorescent channel only) of seedlings treated for 2 h with 10 µM DPI or
529
DMSO for control. Scale bars, 100 µm in all confocal images. g represents gravity vector, Ψ
530
represents water potential gradient. A) Images of unstimulated roots, pre-treated for 2 h with 10
531
µM DPI or DMSO. DHR signal is more intense and penetrates to the deeper root layers due to
532
longer incubation in the dye (5 minutes). Images are representatives of n=23 seedlings. B) DHR
18
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
533
fluorescence intensity of seedlings treated with DPI or DMSO for 2 h, measured at the epidermal
534
layer of the EZ and at the meristematic zone. Error bars represent mean ± SE (3 biological
535
independent experiments, n=23 seedlings in total). *p<0.05, **p<0.01, t-test for independent
536
measurements. C) Seedlings pre-treated with DPI for 2 h were gravistimulated, and show less
537
ROS accumulation and asymmetrical distribution at the EZ. Images shown here are of a more
538
extraneous section of the root, where the differences between DPI-treatment and control are
539
highly detectable. Images are representatives of n = 11 seedlings. D) Seedlings that were
540
hydrostimulated for 2 h on a DPI containing medium showing elimination of the signal from the
541
epidermal layer at the bending region, which became more proximal to the root tip. Images are
542
representatives of n = 20 seedlings.
543
Figure 5: rbohC, but not rbohD, show accelerated hydrotropic bending and lower ROS levels in
544
the root apex. A) Root hydrotropic growth of WT, rbohC and rbohD, 2 h post hydrostimulation.
545
Scale bar, 1 mm. g represents gravity vector, Ψ represents water potential gradient. B) Root
546
curvature kinetics and growth rates. Root curvature was measured at 1 h interval for 7 hours
547
following hydrostimulation. Root growth rate was determined by measuring length at the
548
beginning and at the end of the experiment. Error bars represent mean ± SE (3 biological
549
independent experiments, 10 seedlings each). Statistical difference in root curvature was tested
550
for 2 and 5 h post hydrostimulation. C) Determination of H2O2 content in root apices (1-2 mm
551
from tip) and whole seedlings (D) of WT, rbohD and rbohC, measured by the Amplex red assay
552
(Materials and Methods). The fluorescent reads were normalized to the amount of extracted
553
protein, measured by the Bradford assay. Error bars represent mean ± SD (3 biological repeats
554
with two technical replicates. for root apices, n = 60, for whole seedlings, n = 20). The higher y-
555
scale in C is a result of normalization to ten-fold lower protein level extracted from root apices.
556
In B, C and D) Means with different letters are significantly different (p < 0.05, Tukey HSD
557
adjusted comparisons).
558
Figure 6: Hydrotropism abrogates the gravitropic ROS signal. A) Schematic presentation of the
559
assay applied to test ROS distribution at root tips of hydrostimulated seedlings and a combined
560
gravistimulation with hydrostimulation. B) Roots were hydrostimulated for the indicated times
561
and then gravistimulation for 1 h, stained with Dihydrorhodamin-123 (DHR) and imaged using a
562
confocal microscope (Materials & Methods). Images are presented as pseudo color. Scale bar,
563
100 µm. C) Quantification of DHR fluorescence, measured at the DEZ epidermal layer (200 µm
564
above apex). The data is presented as the ratio between the signal at the concave and the convex
19
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
565
sides of the root. Error bars represent mean ± SE (3 biological independent experiments, n=20).
566
D) Root curvature of 1 h gravistimulated seedlings following hydrostimulation for the indicated
567
times. The 1 h gravitropic curvature following 0, 2, 3 and 4 h hydrosimulation was 14.42o ±
568
1.27, 9.16o ± 0.76, 6.33o ± 0.78 and -3.14o ± 2.03, respectively. Error bars represent mean ± SE
569
(3 biological independent experiments, n=15). Negative value means curvature against the
570
gravity vector direction. In A and B, Ψ and g represent the water potential gradient and gravity
571
vector, respectively. ROS images of hydrostimulated roots for the same indicated times, without
572
gravistimulation are shown in Fig. 1 E. In C and D, letters above bars represent statistically
573
significant differences by Tukey-HSD post hoc-test (P < 0.05).
574
575
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630
Jaffe MJ, Takahashi H, Biro RL (1985) A pea mutant for the study of hydrotropism in roots.
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631
632
Joo JH, Bae YS, Lee JS (2001) Role of Auxin-Induced Reactive Oxygen Species in
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636
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H (2007) A gene essential for hydrotropism in roots. Proc Natl Acad Sci 104: 4724–4729
21
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641
642
643
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660
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Downloaded from on June 15, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
682
683
Sarath G, Hou G, Baird LM, Mitchell RB (2007) Reactive oxygen species, ABA and nitric
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Takahashi N, Yamazaki Y, Kobayashi A, Higashitani A, Takahashi H (2003) Hydrotropism Interacts with Gravitropism by Degrading
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Google Scholar: Author Only Title Only Author and Title
Tsukagoshi H, Busch W, Benfey PN (2010) Transcriptional regulation of ROS controls transition from proliferation to
differentiation in the root. Cell 143: 606-616
Pubmed: Author and Title
CrossRef: Author and Title
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Google Scholar: Author Only Title Only Author
Downloaded
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Winter D, Vinegar B, Nahal H, Ammar R, Wilson G V, Provart NJ (2007) An "Electronic Fluorescent Pictograph" browser for
exploring and analyzing large-scale biological data sets. PLoS One 2: e718-e718
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Xu Y, Xu Q, Huang B (2015) Ascorbic acid mitigation of water stress-inhibition of root growth in association with oxidative defense
in tall fescue (Festuca arundinacea Schreb.). Front Plant Sci 6: 1-14
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
Young LM, Evans ML (1994) Calcium-dependent asymmetric movement of 3H-indole-3-acetic acid across gravistimulated isolated
root caps of maize. Plant Growth Regul 14: 235-242
Pubmed: Author and Title
CrossRef: Author and Title
Google Scholar: Author Only Title Only Author and Title
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